2.3. Ginseng extraction and 1H NMR spectroscopic analysis

Whole ginseng plants collected in the last week of every month were rinsed with cold water and divided into various parts, including main roots, rootlets, rootlet sides, stems, and leaves (Fig. 2). Each part of ginseng plants was then dried for 48 h using a freeze dryer. These freeze-dried ginseng samples were ground into powder using a pestle and mortar under liquid nitrogen. Then, 100 mg of freeze-dried ginseng powder sample was dissolved in a mixture of methanol-d₄ (CD₃OD, 490 μL) and deuterium water (D₂O, 210 μL) containing 0.05% (wt) TSP in a 1.5-mL Eppendorf tube. The mixture was sonicated at room temperature for 20 min and then centrifuged at 13,000 rpm for 15 min at 10 ^oC. The supernatant (600 μL) was then transferred into 5-mm NMR tubes. These extracts were analyzed using a Bruker Avance 700 spectrometer (Bruker Biospin, Rheinstetten, Germany) operating at 700.40 MHz ¹H frequency and a temperature of 298 K coupled with a cryogenic triple-resonance probe and a Bruker automatic injector. Methanol-d₄ in the aliquots provided a field-frequency lock. Signal assignment for representative samples was facilitated by two-dimensional total correlation spectroscopy and heteronuclear single-quantum correlation. In particular, ginsenoside compounds were identified through spiking experiments with their pure chemicals.

Metabolome-wide pattern dependent on seasonal change in main roots of Panax ginseng. Amounts of each metabolite were determined by integration of ¹H NMR peaks. Because each metabolite in ginseng roots had totally different amounts, we considered data transformation to graphically show change patterns of metabolites all together in the current circus plot. Therefore, we took average values of 10 biological replicates for all ginseng plants and then calculated relative proportion of average values within each metabolite. Statistics for the difference of each metabolite are shown in Fig. 7. GABA, γ-aminobutyrate; UDP, uracil diphosphate.